PROJECT SUMMARY/ABSTRACT Non-coding small RNAs (sRNAs), which act swiftly and specifically, are ideal gene regulators in pathogens such as Streptococcus mutans that need to respond to dramatic shifts in environmental conditions within the oral cavity. This ability to tolerate environmental stress is key to its pathogenicity; however, nothing is known about sRNAs that facilitate stress tolerance in S. mutans?the predominant causative agent of dental caries. It is important that we gain better molecular insights into S. mutans because dental caries is one of the most prevalent and costly diseases worldwide, and despite decades of research, the problem is worsening. Our long- term goal is to define the functions of sRNAs in S. mutans and to apply this knowledge to developing novel therapeutic approaches to control tooth decay. Towards attaining this goal, the objective of this application is to identify sRNAs that regulate stress adaptation mechanisms in S. mutans. Based on preliminary data that identified several novel sRNAs in S. mutans, our central hypothesis is that specific subsets of sRNAs are key regulators of different classes of stress tolerance in S. mutans. The rationale for the proposed research is that sRNAs represent a largely untapped reservoir of therapeutic targets that could be exploited to control dental caries as well as many other human diseases caused by imbalance in normal flora. The objective of this study will be accomplished by two Specific Aims: (1) Identify sRNAs that respond to various classes of stress in S. mutans. By combining a novel RNA-seq-based approach with traditional methods such as Northern blot, we will identify sRNAs that are induced or repressed in S. mutans growing under acidic, nutritional, oxidative, and temperature stresses. (2) Define the function of a novel sRNA induced by sugar-phosphate stress. To begin to understand how sRNAs facilitate stress tolerance, we will interrogate the function of SmsR4, a novel sRNA that we recently identified. This sRNA was chosen because it was highly induced in S. mutans exposed to xylitol, a non-metabolizable sugar alcohol that causes sugar-phosphate stress, and is used in dental care products. Almost all studies to date on S. mutans have investigated only the functions of proteins, especially in the context of transcription regulation; hence, the research proposed here is innovative because it will de- part from the status quo to investigate the role of sRNAs in posttranscriptional control of gene expression, thereby stimulating an entirely new area of S. mutans genetic research. This study is significant because it will, for the first time, identify sRNAs in S. mutans that facilitate stress tolerance, which is critical to its cari- ogenicity. This new knowledge is expected to advance our understanding of regulatory circuits in S. mutans. Additionally, because hardly anything is known about this topic in nearly all members of the human flora, the proposed project is likely to uncover basic concepts that could be generally applicable to the maintenance of a healthy flora, thereby improving human health.